Biosensors are analytical tools that can be used to measure the presence of a molecular species in a sample by combining the molecular recognition properties of biological macromolecules with signal transduction mechanisms that couple ligand binding to readily detectable physical changes. Commonly, a biosensor combines a naturally occurring macromolecule (e.g., an enzyme or an antibody), with the generation of a suitable physical signal particular to the molecule in question.
Escherichia coli periplasmic binding proteins are members of a protein superfamily (bacterial periplasmic binding proteins) that has been shown to be suited for the engineering of biosensors (e.g., see, U.S. Pat. Nos. 5,898,069 and 6,277,627). Bacterial periplasmic binding proteins typically comprise two domains linked by a hinge region (Quiocho & Ledvina, Molec. Microbiol. 20:17-25, 1996). The ligand-binding site is typically located at the interface between the two domains. The proteins typically adopt two conformations: a ligand-free open form, and a ligand-bound closed form, which interconvert via a hinge-bending mechanism upon ligand binding. This global, ligand mediated conformational change can be exploited to couple ligand binding to changes in fluorescence intensity by positioning single fluorophores in locations that undergo conformational changes in concert with the global change (e.g., Brune et al., Biochemistry 33:8262-8271, 1994; Gilardi et al., Prot. Eng. 10:479-486, 1997; Gilardi et al., Anal. Chem. 66:3840-3847, 1994; Marvin et al., Proc. Natl. Acad. Sci. USA 94:4366-4371, 1997, Marvin and Hellinga, J. Am. Chem. Soc. 120:7-11, 1998; Tolosa et al., Anal. Biochem. 267:114-120, 1999; Dattelbaum & Lakowicz, Anal. Biochem. 291:89-95, 2001; Marvin & Hellinga, Proc. Natl. Acad. Sci. USA 98:4955 4960, 2001; Salins et al., Anal. Biochem. 294:19-26, 2001).
In biological systems, changes in phosphorylation state and fluctuations in the concentration of inorganic phosphate are associated with a number of important events. Because inorganic phosphate (Pi) is involved in a number of important biological processes, it is often desirable to be able to measure the concentration of Pi and changes in such concentration in biological systems. Phosphate assays, which measure Pi concentration, are useful in a number of diagnostic methods, as well as in research related to the functioning of biological systems.
A number of diseases and conditions present with elevated or depressed levels of serum inorganic phosphate concentration. Moreover, the major energy requirements of the body are fulfilled by deriving energy from alterations in the phosphorylation state of nucleotides. A large number of enzymes of importance to drug discovery consume or produce inorganic phosphate (Pi), either directly or through coupled reactions. These enzymes include protein and lipid phosphatases, ATPases, drug transporters, GTPases, phosphorylases, phosphodiesterases, and prenyl transferases. Additional applications include monitoring of phosphate in clinical samples and in process control within the bioproduction industry. The current standard assay for phosphate quantitation is an absorbance assay based on malachite green, which is sensitive to about 5 μM phosphate, and robust at ˜25 μM phosphate. In addition to limited sensitivity, this assay suffers in that it is absorbance-based, and thus is far from ideal for high throughput screening. A third disadvantage is that malachite green can only be used in an end-point format, precluding kinetic analysis that could be useful for lead characterization and optimization of small molecules. It is desirable to utilize phosphate assays having a rapid response rate, in order to monitor the kinetics of biological and chemical processes which involve the production or consumption of Pi.
Phosphodiesterases cleave phosphodiesters to phospho-monoesters Important classes of phosphodiesterases include those that act upon cyclic nucleotide phosphodiesters (cAMP or cGMP). Present methods to quantitate such activity depend on binding of either the substrate (cNMP) or product (NMP) to a binding partner (an antibody or other reagent) which can be detected by displacement of a fluorescent moiety from that binding partner. Such binding partners can be expensive and can show poor specificity between substrate and product, limiting assay performance. Methods that depend on detecting a decrease in the amount of substrate present are problematic in that low levels of enzyme activity can be difficult to detect. Methods which are part of the invention, described herein, do not suffer from these disadvantages.
Fluorescent and radiometric methods exist to detect kinase activity. However, very few truly “generic” methods exist that can be applied to assay a wide range of kinases. Many kinase assays depend on the use of antibodies that detect specific phosphorylated residues in a substrate, or on the phosphorylation of specific peptide substrates (that may not be optimal for the kinase being assayed). Methods which are part of the invention, described herein, can be used with “native” kinase substrates, do not depend on the use of radioactivity or antibodies, and detect formation of product rather than a decrease in substrate.
There is a broad demand for phosphate detection reagents and assays. These include, for example, both basic and applied biochemists, in pharmaceuticals and academia, as well as high throughput screening facilities. One need among researchers is for sensitive, but also kinetic assays for enzymatic activities. One need among HTS facilities is for sensitive, robust, and miniaturizable assays, preferably, but not necessarily, resistant to compound interference.
Citation or discussion of a reference herein is not to be construed as an admission that such is prior art to the present invention.